Tissue processing apparatus

ABSTRACT

An apparatus for processing a biological sample is provided. The biological sample being arranged on a first planar surface of a carrier, the apparatus having a second planar surface arranged substantially parallel to said first planar surface and at a first distance from said first planar surface. The first planar surface and said second planar surface are arranged at an angle (A) greater than zero degree from the horizontal plane (HP). The apparatus having a supply for supplying an amount of a liquid that is to be applied to said biological sample. The first planar surface and said second planar surface are configured to be arranged at a second distance from each other, said second distance being such that said supplied amount of liquid is distributed over said biological sample when said first planar surface and said second planar surface are brought to said second distance from each other.

PRIORITY CLAIM

This is a continuation of application Ser. No. 12/747,023, filed Sep. 3,2010, which is a U.S. National Phase Application based onPCT/DK2008/000430, filed Dec. 9, 2008, which claims the benefit of U.S.Provisional Application No. 61/012,481, filed Dec. 10, 2007, both ofwhich are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to processing of a biological sample fore.g. histological and cytological examination. Especially, the presentinvention relates to the processing of a biological sample, for examplea thin tissue section, using a small quantity of a processing liquid.

BACKGROUND OF THE INVENTION

Sample processing in immunohistochemical (“IHC”) applications, forexample, and in other chemical and biological analyses may involve oneor a number of various processing sequences or treatment protocols aspart of an analysis of one or more samples. Typically, such treatmentprotocols are defined by organizations or individuals requestinganalysis, such as pathologists or histologists attached to a hospital,and may be further defined by the dictates of a particular analysis tobe performed.

A fluorescence In-situ hybridization (FISH) procedure is traditionally atwo days manual procedure. Attempts have been made to automate parts ofthe procedure in order to shorten the processing procedure and to reducethe number of manual steps. For example, the first day pre-treatmentprocedure has been automated with an instrument VP2000™ (Vysis, AbbottMolecular), in which instrument a robot moves slides from one jar toanother. However, the problem so far has been to combine thepre-treatment steps of the first day and the washing steps of the secondday with the strict physical and environmental requirements of thedenaturation and hybridization steps in between. In these steps it ispreferred to use small volumes of processing liquids and provide aprecise control of the humidity in the processing chamber surroundingthe processed tissue section, and to provide controlled heating andcooling in order to obtain consistent FISH results.

Automated IHC and ISH staining instruments have been introduced byVentana Medical Systems Inc. (BenchMark™ and Discovery™) andVisionBiosystem (Bond™). A drawback with these instruments is that theyonly provide a fixed processing volume, i.e. the processing chamber isof a fixed volume. The processing chamber volume in the instrument beingat least 100 micro litres.

The BenchMark™ instrument, having a capacity to process 30 slides, needsto cover the tissue section to be processed and the applied processingliquid with oil in order to reduce evaporation of the processing liquid.If not covered by oil, the evaporation of processing liquid willdeteriorate the processing result.

The Bond™ instrument, having a capacity to process 30 slides, has asmall processing chamber which is clamped over each tissue section andeach carrier. By clamping the processing chamber over each carrier, anindividual staining cavity is created.

Some of the drawbacks with prior art instruments are that they requirerelatively large volumes, about 150-200 micro litres, of processingliquid, that they do not provide as good results as manual processing,and that they do not provide a variable volume of processing liquid tobe used by providing a processing chamber having a variable volume.

An aim of the present invention is to solve these and other problems anddrawbacks with the prior art system.

For example, an object of the present invention is to provide processingof a biological sample arranged on a carrier using a small quantity ofprocessing liquid.

Another object of the present invention is to provide processing of abiological sample arranged on a non-horizontal carrier.

Yet another object of the present invention is to provide a processingapparatus for processing of a biological sample arranged on a carrier,the processing apparatus being configured to provide a variable volumefor processing liquid.

Another object is to provide automated processing of a biological samplearranged on a carrier using a small quantity of processing liquid.

SUMMARY OF THE INVENTION

The present invention concerns molecular pathology, i.e. the examinationat a molecular level of the DNA; mRNA, and proteins that cause or areotherwise associated with disease. The present invention relates toprocessing of a biological sample for e.g. histological and cytologicalexamination. Especially the present invention relates to the processingof a thin biological sample, e.g. a tissue section, using a smallquantity of a processing liquid.

In particular, the invention relates to processing, treating and/orstaining of at least one biological sample, e.g. a tissue section,accommodated on a carrier as well as to the control of the humidity andtemperature during the processing and/or treating and/or staining.

It should be understood that the present invention may be used in thefields of cytology and histology, molecular biology, biochemistry,immunology, microbiology, and cell biology. In particular, the inventionrelates to the fields of molecular cytogenetics andimmunohistochemistry, for processing biological samples inimmunohistochemistry (IHC), in-situ hybridization (ISH), fluorescentin-situ hybridization (FISH), chromogenic in-situ hybridization (CISH),special stains (SS), silver in-situ hybridization (SISH), microarrays(tissue, protein, RNA, DNA, PNA, LNA, etc.) as well as other chemicaland/or biological applications.

The inventive processing apparatus provides a variable processingvolume, whereby different biological samples can be processed usingdifferent volumes of processing liquids, e.g. different volumes ofreagent can be used for different biological samples.

Advantages of Embodiments of the Present Invention

Embodiments of the present invention comprise one or more of thefollowing features:

Feature Advantages Explanation/Comment Angle A passive draining gravity(different from horizontal) physical container Contained liquid variablevolume Different volumes of An angle and gravidity processing liquid maybe combined can be used to used hold liquid contained in “open chamber”system smaller footprint of Compared to horizontal apparatus slideposition Faster drying Liquid dries faster by running down a slide withan angle during drying during heating No coffee ring effect Due to theangle Minimize air bubbles Air can easier move away due to angleMinimize evaporation The higher angle combined with the blocks areareduces the surface of which evaporation can occur liquid/humidityreservoir An angle can by gravity hold liquid for humidity controlseparated from reagents in a simple manner Geometry of Round shapedpiston The bottom of a piston can block be shaped such that it fits to aslide. It can also cover a portion of a slide or the whole slide Twosided block to two Notice that the angle of slides or more which theblock lowered to the slide is not perpendicular as the one slide blockSquare Cover a portion of slide or cover whole slide Rectangular Cover aportion of slide or cover whole slide Diamante As batch and/or singlemode Block(s) system geometry They can e.g. be placed in carousel,linear, spiral etc.. Block Variable volume Provide a span of a minimumfactor or 1000 in volume, e.g. from 20 micro litres to 20 millilitres,or even more Spread of reagent Bottom can be flat or modified to fit asa “cover glass” for e.g. a portion of the slide surface area Controlleddifferent volumes By moving block in one dimensional direction volumescan vary from e.g. 10 micro litres to 100 millilitres Efficient mixingIncrease kinetic speed, mix different reagents under/on/above/slide nocover glass Block can also contain the function of a traditional coverglass no cover glass sealant batch mode One block made such that itcovers multiple slides, e.g. 12 single mode One block made such that itcovers one slide “Humidity” reservoir For example in block and/or inchamber fast multi processing Simple design Slide position block orplaten or both Controlled environment (humidity/temp) No oil orequivalent for sealing necessary Fast temperature ramping due to smallvolumes Many possibilities for above, through wall side, reagentaddition through platen, capillary forces, through block, out side block(pulling slide out) Liquid reservoir Can be build into block, wall orplaten Semi closed changed to open container by removing the block Theblock can move by one dimension movements Automated movement of Easilyautomated block Nose barrier Might have a form for O- ring to assistsealing (so far not required) One brick can in principle perform alltypes of processes. However, it might be preferred to optimize the blockto different types of processes such as e.g. IHC and FISH especially inlight of the potential long hybridisation time (14-20 h). Made of a poorheat Fast ramp times of small conductor liquid volumes Slide position Inplaten (bottom plate) Below surface, at surface or above surface ofplaten In block Below surface, at surface or above surface of platenSlide facing down Reagent e.g. through platen can be pre-heated byplaten Two slides at same time Use less reagent, smaller (same reagente.g. Her2) instrument Slide may be “tilted” X, Y, Z dimension All stepswhile slide is in one Capable of performing all position reagentprocessing steps Variable volume in batch Batch instrument mode ISH,FISH, CISH, SISH, SS, IHC etc Combined batch and single Block can bedesigned to By e.g. building in a small mode do both wall, going intothe block, between the slide positions both batch and single mode can becombined Multi flexible system Can potentially run with or without arack holding the slides Mixing In chamber by moving block e.g. 1 mm upand down Built in reagent mixing station in chamber Increased reactionspeed on slide with active mixing and temperature control Mixconcentrate of e.g. Ab to RTU on side, of diverse buffer concentrate toRTU etc. (RTU = Ready-to-use liquid) Mixing during incubation Canpotential decrease incubation times and make it possible to use smallervolumes than a static incubation. Mixing large and small By moving thebrick a very By having an angle there volumes efficient mixing can bewill be no “dead” spot on made. mixing of small volumes as gravitytogether with capillary forces see to that the liquid will move to thebottom part of the slide and thereby secures a homogenous mixing (andwashing) in contrast to horizontal. Large wash volumes e.g. 4 mL Thewhole chamber is in Movement of brick will use increase wash effiencySmall wash volumes e.g. “Coverslide” of brick is in Small movement ofbrick 150 uL use will increase wash efficiency Circulation of liquidCirculation of liquid can e.g. be performed by outlet through overflowdrain and back again through the bottom inlet. Reagent Assist release ofslide after inlet/outlet e.g. o/n hybridization by inlet of wash bufferthrough bottom before raising block Possibility of small volumes withsemi-closed (read closed) block position Inlet channel through chamberwall Chamber Overflow drain Can be used to e.g. separate organic waste,secure against overflow, used in washing steps etc. Inlet Outlet Guiderail for block Chamber sealant, e.g. by o- Around chamber and/or Securefree movement rings block with potential material expansion orcontraction at diverse temperatures Reservoir for liquid to secure highrelative humidity

An embodiment includes an apparatus for processing a biological sample,said biological sample being arranged on a first planar surface of acarrier, said apparatus comprising:

-   -   a second planar surface at a first distance from said first        planar surface, said first planar surface is arranged at an        angle (A) greater than zero degree from the horizontal plane        (HP);    -   a liquid supply for supplying an amount of a liquid that is to        be applied to said biological sample;    -   wherein said first planar surface and said second planar surface        are configured to be arranged substantially parallel at a second        distance from each other, said second distance being different        from said first distance and being such that said supplied        amount of liquid is distributed over said biological sample when        said first planar surface and said second planar surface are        brought to said second distance from each other.

In an embodiment the second planar surface is arranged substantiallyparallel to said first planar surface and at a first distance from saidfirst planar surface, said first planar surface and said second planarsurface are arranged at an angle (A) greater than zero degree from thehorizontal plane (HP).

A simple construction is needed to move the second planar surface fromthe first position to the second position when the first and secondsurfaces are parallel in both positions.

In embodiments, the sample carrier is arranged at an angle A from thehorizontal plane, whereby passive draining of liquid from the sample isprovided. The passive draining is due to gravity and/or capillaryforces. An advantage by such an arrangement is that duringdrying/dehydration/baking of specimen, liquid is removed faster due tothe combined effect of the heating and the passive draining thancompared with sample processing apparatuses having sample carriersarranged parallel to the horizontal plane.

Arranging the sample carrier at an angle A also provides that differentvolumes of processing liquid may be used. The liquid can be held in acapillary field when using small volume and when using larger volume thechamber walls and block act as a chamber and retain the liquid. Such aprocessing chamber can suitably function as a humidity chamber.

Yet another advantage with providing the sample carrier at an angle A isthat a smaller footprint of the apparatus may be achieved than if thesample carrier was arranged parallel with the horizontal plane.

Another advantage is that air bubbles trapped in the processing liquidbetween the sample carrier and the block more easily may escape from theprocessing liquid than if the sample carrier was arranged parallel tothe horizontal plane.

Yet another advantage is that the angle A in combination with the areaof the block reduces the surface from which evaporation can occur,whereby smaller volumes of processing liquid may be used than if theevaporation surface was larger.

A further advantage is that the angle A and the gravity can hold liquidfor humidity control separated from reagents and specimen in a simplemanner by draining condensed liquid back to a humidity reservoir.Thereby, the risk of mixing the reagent used in the specimen processingwith the liquid used for humidity control is reduced.

In embodiments of the invention, the block may be configured as a roundshaped piston. The bottom of the piston may be shaped such that it fitsto a sample arranged on sample carrier. The bottom of the piston may bedesigned to cover a portion of a sample carrier or the whole samplecarrier.

In embodiments, the block is a two-sided block each of which side isconfigured to fit to a sample carrier, whereby two samples potentiallycan be processed simultaneously.

In embodiments having a block with e.g. a square or rectangular shape,the block should be designed to cover a portion of the sample carrier orto cover the entire sample carrier. Embodiments with a diamante shapedblock provides for batch and/or single mode operation.

However, it should be understood that the block may have anothersuitable design and dimension than those described herein.

Embodiments of the invention may also comprise one or more blocksarranged in a carousel arrangement, a linear arrangement, or spiralarrangement etc. In some embodiments one or more of the arrangements canbe stacked.

An advantage with the use of a block is that a variable processingvolume can be provided. It is for example possible to provide a span ofa minimum factor of 1000 in volume, e.g. from 20 micro litres to 20millilitres, or even more. Further, by moving block in e.g. aone-dimensional direction, the processing volume can vary from e.g. 10micro litres to 100 millilitres. Furthermore, by means of the block, theprocessing liquid, e.g. reagents, can be spread over the sample to beprocessed. The bottom of the block can be flat or modified to fit as a“cover glass” over e.g. a portion of the sample carrier surface area.

Yet another advantage with a movable block is that an efficient mixingof processing fluids can be provided by moving the block and therebyproviding a mixing movement in the fluids.

Yet another advantage with the block is that no cover glass may beneeded, the block can also contain the function of a traditional coverglass. A further advantage with the block of the present invention isthat no cover glass sealant is required. Further, depending of thedesign of the block, samples can be processed in batch mode by a blockcovering multiple samples or in single mode by a block covering a singlesample only. Another advantage is that the block can be configured tocomprise a humidity reservoir, whereby a desired relative humidity maybe provided at the sample to be processed. The humidity reservoir canalso be placed in the platen.

In embodiments, the block provides a controlled environment, i.e. acontrolled humidity and/or a controlled temperature.

In embodiments, no oil or equivalent for sealing may be necessary.

Embodiments of the invention provide a fast temperature ramping due tosmall volumes of the processing liquids used.

Embodiments of the invention may also provide different possibilitiesfor reagent supply. The reagent may for example be supplied from above,through the wall side, through the platen, by means of capillary forces,through the block, from the outside of the block by pulling the slideout from the apparatus, etc.

Embodiments of the invention may also provide a liquid reservoir, whichmay be arranged at the block, the wall or the platen (the bottom plateof the processing chamber).

In embodiments, the processing apparatus is configured with a semiclosed chamber being a closed chamber when the block is in a processingposition.

In embodiments, the block may be moved by one-dimensional movements.

In embodiments, the block may be moved by two-dimensional movements.

Some embodiments provide automated movement of the block.

In embodiments, the apparatus and the block may be configured to performall types of sample processing. However, it might be preferred tooptimize the block to different types of sample processing, such as e.g.IHC and ISH, or processing steps, such as baking, target retrieval,deparaffination, stringency wash, cover slipping, staining, enzymatictreatment, etc. In the context of the present invention the sampleprocessing is to be understood as the active process of qualifying orquantifying the presence of a specific compound.

In embodiments, the block is configured of a poor heat conductingmaterial, whereby the block does not or to a limited extent conduct heatsupplied to the processing liquid. Thus, the temperature of theprocessing liquid may be changed quickly within a short ramp time.

In embodiments of the present invention, the sample may be arranged atthe bottom plate of the processing chamber. Further, the sample may bearranged below the surface of, at the surface of or above the surface ofthe bottom plate.

In other embodiments, the sample is arranged at the block. Further, thesample may be arranged below the surface of, at the surface or above thesurface of the block.

In some embodiments, the sample on the carrier is facing down, i.e. thesample is arranged at the block facing the bottom plate of theprocessing chamber, whereby processing liquid, e.g. a reagent, suppliedthrough the bottom plate can be pre-heated by the bottom plate withoutthe sample being pre-heated by the bottom plate.

In some embodiments, two slides are processed at the same time using thesame processing liquid, e.g. a Her2 reagent, whereby less processingliquid may be used than if the samples were processed sequentially.

Further, the sample carrier may be arranged in a “tilted” position, e.g.the sample carrier may be titled in the X, Y, or Z direction. If thesample carrier is arranged at an angle A from the horizontal plane, thefootprint of the apparatus may be less than if the sample carrier wasarranged parallel with the horizontal plane.

In embodiments, all processing steps are accomplished while the samplecarrier is in one position.

In embodiments of the invention, one or more sample carriers arearranged in a rack, whereby the one or more sample carriers are handledby means of the rack, i.e. the one or more sample carriers may beinserted in or removed from the sample processing apparatus by insertingor removing the rack in or from the processing apparatus.

In embodiments of the invention, mixing is provided in the processingchamber by moving the block e.g. about 1 mm up and down. Embodiments mayalso comprise a built in mixing station for processing liquid, e.g. areagent mixing station in the apparatus, e.g. in the processing chamber.By means of active mixing and possible temperature control, an increasedreaction speed on the sample is provided.

Embodiments may comprise mixing of concentrates e.g. antibodies (Ab) toready-to-use reagents (RTU) on site or mixing of different bufferconcentrates to RTU liquids, etc.

Embodiments may provide mixing during incubation, whereby incubationtimes may be decreased and smaller volumes of processing liquids, e.g.reagents, may be used as compared to the case of static incubation.

Embodiments may provide mixing of large and small liquid volumes. Bymoving the block, e.g. up and down, a very efficient mixing can be made.By having the sample carrier arranged at an angle to the horizontalplane there will be no “dead” spot when mixing small volumes sincegravity together with capillary forces provide that the liquid will moveto the bottom part of the sample carrier and thereby secure a homogenousmixing (and washing) in contrast to the case when the sample carrier isarranged parallel to the horizontal plane.

Embodiments of the invention providing movement of the block may alsoprovide an efficient washing.

In embodiments of the invention, processing liquid is circulated throughthe processing chamber. For example, circulation of liquid may beperformed by circulating the liquid out of a chamber outlet e.g. throughan overflow drain and back again through an inlet, e.g. a bottom inlet,of the processing chamber.

In embodiments, the invention can be used to create a capillary field.For example the filling of the chamber/carrier partly or fully withliquid with the block in a first position, followed by moving the blockto a second position creates a capillary field when removing thenon-capillary bound liquid from the chamber by e.g. a reagent outlet.Another way is to purge liquid through the chamber when the block is inthe second position, creating a capillary field.

In embodiments, the processing chamber is provided with an overflowdrain that may be used to e.g. separate organic waste, secure againstoverflow, used in washing steps etc. The processing chamber may also beprovided with an inlet and an outlet. Further, in embodiments, thechamber may be provided with a guide rail for the block. In embodiments,the chamber may be provided with a chamber sealant, e.g. one or moreo-rings. The chamber sealant may be arranged around the chamber and/orthe block. In embodiments, secure free movement of the block takingpotential material expansion or contraction at diverse temperatures intoconsideration is provided.

In embodiments, the processing chamber comprises a reservoir for liquidto secure high relative humidity within the processing chamber.

In embodiments, the block may be designed to provide both a batch modeand a single mode of operation. For example, in such embodiments, theblock may be provided with one or more protrusions, e.g. parallel wallsections, separating one or more sample carriers from each other whenthe block is arranged in a processing position.

Further embodiments include:

an apparatus wherein said supply means is configured to supply saidamount of said liquid to a first part of said biological sample whensaid second planar surface is at said first distance from said firstplanar surface, and wherein said applied amount of liquid is distributedover a second part of said biological sample when said first planarsurface and said second planar surface are brought to said seconddistance from each other, said second part of said biological samplehaving a larger surface area than said first part of said biologicalsample.

an apparatus wherein said supply means is configured to supply saidamount of said liquid to said first planar surface or to a first part ofsaid biological sample when said second planar surface is at said seconddistance from said first planar surface, and wherein said applied amountof liquid is distributed over said biological sample due to capillaryaction.

an apparatus wherein said supply means is configured to supply saidamount of said liquid to said second planar surface, and wherein saidsupplied amount of liquid is distributed over said biological samplewhen said first planar surface and said second planar surface are atsaid second distance from each other.

an apparatus comprising a processing chamber having a first structureconfigured as a container having a bottom plate and walls and a secondstructure configured as a block, wherein said block is dimensioned tofit the opening of the container. Such a processing chamber can suitablyfunction as a humidity chamber.

an apparatus wherein said bottom plate of said first structure isconfigured to support said carrier having said first planar surface.

an apparatus wherein said second structure is shaped as a block having asurface constituting said second planar surface.

an apparatus wherein said first structure comprising said second planarsurface.

an apparatus wherein said second structure is configured to hold saidcarrier having said first planar surface.

an apparatus wherein said second planar surface comprises a planarelevation arranged to cover at least a part of said biological samplewhen said second planar surface is at said second distance from saidfirst planar surface.

an apparatus wherein said elevation has the shape of a cover glass.

an apparatus wherein a sealing is arranged at the outer boundary of saidplanar elevation, whereby said applied amount of liquid is retainedwithin a space defined by said sealing, said planar elevation, and saidpart of said biological sample.

an apparatus comprising a control unit configured to control the supplyof liquid from said supply means.

an apparatus comprising a distance changing means for changing thedistance between said first planar surface of said carrier and saidsecond planar surface.

an apparatus wherein said distance changing means is an automaticdistance changing means for automatically changing the distance betweensaid first planar surface of said carrier and said second planarsurface.

an apparatus comprising a control unit configured to control saiddistance changing means.

an apparatus wherein said carrier having said first planar surface is amicroscope slide.

an apparatus wherein said angle (A) is larger than 0 degrees and smallerthan or equal to 90 degrees.

an apparatus wherein said angle (A) is between 30 degrees and 60degrees.

an apparatus wherein said angle (A) is approximately 45 degrees.

an apparatus wherein said liquid is a reagent or a buffer solution.

an apparatus comprising a lid configured to be arranged at said firststructure to enclose said carrier and said second structure duringprocessing of said biological sample arranged on said first planarsurface.

an apparatus comprising a reservoir arranged at surface of said lid thatfaces said second structure, said reservoir being configured to controlthe humidity within the processing chamber during processing of saidbiological sample.

an apparatus wherein said reservoir is configured as an elongatedreservoir comprising an absorbing material.

a processing chamber for processing a biological sample arranged on afirst planar surface of a carrier, said processing chamber comprising:

-   -   a second planar surface arranged substantially parallel to said        first planar surface and at a first distance from said first        planar surface, said first planar surface and said second planar        surface are arranged at an angle (A) from a horizontal plane        (HP) during processing of said biological sample.

One or more of the following methods and steps can be performed by usingthe apparatus of the invention;

-   -   Baking of e.g. formaldehyde fixed paraffin embedded tissue    -   No reformatting, i.e. the sample carriers need not to be moved        between processing steps—same position    -   Fixation    -   Dehydration—e.g. by heat after wash/rinse with e.g. water or        dehydration with ethanol    -   Deparaffination    -   Target retrieval/pre-treatment    -   Wash steps—Static (add liquid, no movement of brick) or dynamic        (brick movement or liquid flow) and flush    -   Digestive treatment—Controlled temperature with potential low        volume and controlled environment (humidity) secure more        standardized digestion    -   Denaturation    -   Hybridization    -   Stringency wash    -   Mounting and coverslipping    -   H&E staining    -   Special stains    -   IHC staining    -   ISH (in-situ-hybridization)    -   Counter staining

BRIEF DESCRIPTION OF THE DRAWINGS

The objects, advantages and effects as well as features of the presentinvention will be more readily understood from the following detaileddescription of embodiments of the invention, when read together with theaccompanying drawings, in which:

FIGS. 1 a and 1 b schematically show an embodiment of an apparatus forprocessing a biological sample according to the invention;

FIG. 2 schematically shows a cross-sectional view of an embodiment of anapparatus for processing a biological sample according to the invention;

FIGS. 3 a and 3 b schematically show an embodiment of an apparatus forprocessing a biological sample, the apparatus comprising a lid.

FIG. 4 schematically shows an embodiment of a carrier for a biologicalsample to be processed;

FIGS. 5 a and 5 b schematically show an embodiment of a carrier for abiological sample with an amount of a processing liquid applied;

FIGS. 6 a-6 d schematically show different embodiments of a block for aprocessing apparatus according to the invention;

FIG. 7 schematically shows an embodiment of the processing apparatusaccording to the invention;

FIG. 8 schematically shows an embodiment of the processing apparatusaccording to the invention;

FIGS. 8 a, 8 b, 8 c, and 8 d schematically show how processing liquidmay be supplied in embodiments of the processing apparatus according tothe invention;

FIG. 9 schematically shows an embodiment of the processing apparatusaccording to the invention;

FIGS. 10 a and 10 b schematically show an embodiment of a processingapparatus for processing two biological samples arranged on twocarriers;

FIGS. 11 a-11 c schematically show embodiments of multi-processingapparatuses for processing several biological samples arranged onseveral carriers;

FIG. 12 schematically shows parts of an embodiment of the processingapparatus according to the invention;

FIGS. 13-15 schematically show parts of an embodiment of the processingapparatus according to the invention;

FIG. 16 schematically shows an embodiment of a block;

FIG. 17 schematically shows an embodiment of an inventive system;

FIGS. 18 a, 18 b and 18 c show schematically embodiments of a block;

FIG. 19 schematically shows an embodiment of an apparatus for processinga biological sample according to the invention, in the embodiment one ofthe side walls being removed;

DETAILED DESCRIPTION OF THE INVENTION

While the invention covers various modifications and alternativemethods, apparatuses and systems, embodiments of the invention are shownin the drawings and will hereinafter be described in detail. However, itis to be understood that the specific description and drawings are notintended to limit the invention to the specific forms disclosed. On thecontrary, the scope of the claimed invention is intended to include allmodifications and alternative constructions thereof falling within thespirit and scope of the invention as expressed in the appended claims tothe full range of their equivalents.

FIGS. 1 a and 1 b schematically illustrate an embodiment of an apparatus100 for processing a biological sample 101 mounted on a first planarsurface 102 of a carrier 103.

The biological sample 101 is preferably a thin tissue section of atissue sample, e.g. a human tissue sample.

The carrier 103 may for example be a microscope slide 103 as illustratedin e.g. FIGS. 1 a and 4, but it should be understood that the carriermay have another shape than rectangular. The carrier 103 comprises afirst planar surface 102 carrying the biological sample 101. The carrier103 comprises further an area 115 for information. For example the area115 may be a read and/or write area for reading and/or writinginformation. The area 115 may for example carry an identification tag,such as a bar code label, identifying the biological sample 101. Thearea 115 may also carry information relating to the processing of thebiological sample 101, which may be read by a reader (not shown) such asa bar code reader.

The area 115 is preferably arranged at an upper part 103U of the carrier103. The upper part 103U of the carrier 103 is an upper part of thecarrier 103 when the carrier is in a tilted position, as illustrated inFIGS. 1 and 2. In this tilted position a lower part 103L of the carrier103 is arranged in a lower part of the apparatus when the carrier is inthe tilted position.

The carrier 103, a bottom plate 111 configured to support the carrier103, and/or block 110 may comprise a groove (not shown) configured tofit a finger tip to ease the removal of the carrier after the processingprocedure has finished. Alternatively, a part of the carrier comprisingthe area 115 could protrude from the processing chamber as is shown inFIG. 2. In embodiments this part of the carrier protruding from theprocessing chamber may be removably attached to a carrier rack to easeremoval of the carrier from the processing chamber and to facilitatefurther processing. In embodiments providing processing of multiplebiological samples arranged on multiple carriers a multiple carrier rackis especially advantageous.

As illustrated in FIGS. 1 a and 1 b, the processing apparatus 100comprises a processing chamber 108 comprising a first structure 109 anda second structure 110. The first structure 109 comprises a bottom plate111 and side walls 112 a, 112 b, 112 c, and the second structure 110comprises a block 110.

In FIGS. 1 a and 2, the first structure 109 and the second structure 110are illustrated as having a triangular cross-section. However, it shouldbe understood that this is only for illustrative purpose and that thefirst structure 109 and the second structure 110 may take other shapes,some of which shapes are shown in FIGS. 6 a-6 d. In some embodimentsthere is defined a small confined volume 120 at the biological sample101 in order to provide minimal usage of process liquids.

In the embodiment shown in FIGS. 1 a and 2, the confined volume 120 isdefined by the biological sample 101 on the first planar surface 102 ofthe carrier 103, the inner sides of walls 112 a, 112 b, 112 c, and by asecond planar surface 104 of the block 110.

However, it should be understood that the confined volume 120 may bedefined by the biological sample 101 on the first planar surface 102 ofthe carrier 103, the carrier 103 being mounted on the second structure110 also called block, the inner sides of walls 112 a, 112 b, 112 c, andthe bottom plate 111.

Components and parts of the present invention defining the compartmenthaving the confined volume 120 can be controlled with very highaccuracy, whereby the biological sample 101 can be surrounded by adesired amount of liquid, e.g. processing liquid, during processingthereof.

FIG. 2 illustrates a cross-sectional view of an embodiment of theprocessing apparatus 100 when the block 110 is in a processing position,i.e. when the block 110 is in its lowermost position. This could also beexplained as the block 110 is in a position where the distance betweenthe biological sample 101 on the first planar surface 102 of the carrier103 and the second planar surface 104 of the block 110 is as small aspossible, i.e. the biological sample 101 is touching or almost touchingthe second planar surface 104 of the block 110.

As illustrated in FIGS. 1 and 2, the second planar surface 104 isarranged substantially parallel to said first planar surface 102.Further, the first planar surface 102 and the second planar surface 104are arranged at an angle A from the horizontal plane HP. The angle A ispreferably greater than zero degree, between 5 and 90 degrees, or morepreferably between 30 and 90 degrees. In embodiments, the angle A is 45degrees and in some embodiments the angle is about 20 degrees.

It should be understood that the angle A could be defined as greaterthan zero degree in the x, y, or z plane. Zero degree is defined as theangle A the carrier has to the horizontal plane (x plane) HP when thecarrier is parallel to the horizontal plane HP.

Further, the angle A of the bottom plate and of the carrier provides afast and easy removal of processing liquids. It should be understoodthat processing liquids may refer to all liquids applied to thebiological sample and the carrier, such as washing solution(s),buffer(s), detection reagent(s), deparaffination reagent(s), specialstain reagent(s), probe reagent(s), antibody reagent(s), etc. known to aperson skilled in the art.

In embodiments of the invention, a seal e.g. a rubber seal such as ano-ring may be provided in order to seal the processing chamber.

As illustrated in FIGS. 6 a-6 d, the block 110 may have differentshapes. For example, it may be rectangular, triangular, polygonal,convex, V-shape, \-shaped or diamond shaped. The block may be solid andit may be made of a combination of different materials.

In order to secure a high relative humidity within the processingchamber 108 during e.g. a hybridization step and thereby avoiding thebiological sample 101 to be dried out, embodiments of the invention areconfigured to retain a small volume of liquid in the bottom of theprocessing chamber 108.

In embodiments, the apparatus 100 may be designed to provide a reservoir121, i.e. a humidity chamber 121, for humidity control. The block 110may comprise a recess (cf. FIGS. 14 and 16) in order to provide saidreservoir 121. The reservoir can also be placed in the bottom plate orthe walls (cf. FIG. 7). The humidity chamber 121 is configured tocontrol the relative humidity within the processing chamber 108 duringprocessing of the biological sample 101.

The processing liquid may be removed from the bottom of the processingchamber 108 by means of an outlet, e.g. a drain 122, arranged in abottom part. The drain 122 being controlled by means of a valve 123 e.g.a magnetic valve. When the magnetic valve is closed, the bottom of theprocessing chamber 108 is sealed. Draining of processing liquid may beaccomplished by the gravitation force and/or capillary force acting onthe processing liquid. This can be seen in FIG. 7.

In embodiments, the processing chamber 108 is provided with one or morepikes 127 in a bottom part thereof, e.g. at a liquid outlet. Forexample, the pikes 127 may be arranged in a bottom part of the block,cf. FIG. 14, and/or in the bottom plate 111, cf. FIG. 1. The pike 127being configured to destroy and remove possible liquid bubbles occurringwhen the liquid is removed from the sample and the sample carrier.

In the embodiment shown in FIG. 1, the bottom plate 111 comprises arecess or a groove 113 for supporting the carrier 103. The recess 113being designed to fit the dimensions of the carrier 103. However, itshould be understood that the bottom plate 111 may comprise an elevation116 comprising said recess 113.

Further, it should be understood that the bottom plate 111 may supportthe carrier 103 without comprising a recess or a groove 113.Furthermore, the bottom plate 111 may comprise an elevation 116 forsupporting the carrier 103.

The bottom plate, sometimes also called platen, 111 should be of anefficient thermal conducting material when comprising a heating and/orcooling element(-s) 124, cf. FIG. 8. The temperature of the biologicalsample 101 on the carrier 103 may thereby be easily and efficientlycontrolled by either providing heating or cooling by means of a heatingand/or a cooling element 124 arranged in the bottom plate 111.

In embodiments providing heating and/or cooling from the bottom plate111, the block 110 may comprise an ineffective heat conducting material.The material may be Teflon™ coated to repel the applied processingliquid and to assist spreading out the applied processing liquid.

It should be understood that the heating and/or cooling element inembodiments may be arranged to supply heating and/or cooling from thebiological sample facing side alone or in combination with the heatingand/or cooling from the carrier facing side.

In embodiments providing heating and/or cooling from the biologicalsample facing side, the heating and/or cooling element may be arrangedin the block.

In embodiments, the heating element 124 comprises heat wires or aPeltier element or a microwave element supplying in a controlled mannerthe heat required for the processing. In the ISH, IHC and special stainsprocedures of today, the temperature has to be varied considerably, e.g.between ambient temperature and 99 degrees Celsius.

In embodiments comprising a cooling element in e.g. the bottom plate111, a channel (not shown) may be arranged in e.g. the bottom plate 111to provide an efficient cooling of the carrier 103 and the biologicalsample 101 by ventilation by means of a fan (not shown). The fan may usethe ambient air to cool the tissue section and carrier from a hightemperature to a lower temperature. In embodiments, this may be done byletting ambient air pass cooling ribs (not shown) to cool down thebottom plate and thereby also the carrier 103, the tissue section 101and the processing liquid applied. Similarly cooling can be performed byusing water or a coolant instead of air. Due to the possibility of verysmall liquid volumes for liquids applied to the biological sample, fasttemperature changes can be conducted.

FIGS. 8 a, 8 b, 8 c, and 8 d schematically show the supply of processingliquid to one or more samples according to embodiments of the inventiveprocessing apparatus.

FIG. 8 a shows that processing liquid, illustrated as a droplet 105, maybe supplied by means of a supply means 131 arranged in the block 110,may be supplied at an upper part 103U of the sample carrier 103, and/ormay be supplied by means of an inlet 122 arranged at the bottom part ofthe processing apparatus. Said inlet 122 can also function as a drain.According to embodiments, the block 110 is in a lower position, e.g. ina processing position, when the processing liquid is supplied.

FIG. 8 b shows that processing liquid may be supplied to the sample bymeans of a supply means 131 when the block 110 is in an upper position.

FIG. 8 c shows an embodiment in which processing liquid is supplied bymeans of a supply means 131 arranged in the bottom plate 111 of theprocessing apparatus.

FIG. 8 d shows an embodiment in which processing liquid is supplied bymeans of a supply means 131 arranged in a side wall of the processingapparatus, e.g. the side wall 112 b of the first structure 109. Itshould be understood that, in embodiments, the supply through the sidewall may be accomplished in an upper part, a middle part and/or a lowerpart of the processing apparatus.

FIG. 9 shows an embodiment of the processing apparatus comprising tubing134, 128 connecting a liquid source 126 and a liquid waste 125 to theprocessing chamber 108. In the embodiment, the tubings are connected toan inlet/outlet 122 arranged at the bottom part of the processingchamber 108. The inlet/outlet 122 could be the drain 122 mentionedabove.

FIG. 2 shows an optional pushing and pulling mechanism 117 for pushingand pulling the block 110 in the directions indicated by the arrow. Thispushing and pulling mechanism 117 may be controlled by means of acontroller to provide automatic movement of the block.

The movement of the block may be controlled mechanically by means of thepushing and pulling mechanism 117. In an alternative embodiment, theblock could be arranged to float on the processing liquid applied, i.e.the block may be configured of a material selected so that the block canfloat on the processing liquid applied. In such an embodiment, the blockcould be held in position by e.g. two vertical rods that secure a onedimensional movement along the z-axis for a carrier in the x/y plane andthat provides a maximum float height corresponding to said secondposition, i.e. of e.g. 3 mm.

In embodiments, the block is touching or very close to touching thecarrier surface holding the tissue section when the block is in aprocessing position. Thereby, processing liquid, e.g. probe buffer orreagent, applied to the biological sample will be spread out by theclose physical proximity between the block and the carrier.

In embodiments, the surface of the block spreading out the appliedprocessing liquid is a homogenous and even surface. A pattern texture ofthe surface of the block may be provided. The material of the surface ofthe block may be Teflon™, whereby the small volume of applied processingliquid is more easily spread out over the biological sample.

In a first distance, the block will be at a distance from the carrier.In embodiments, the distance may be 1-3 mm or more. In this positionprocess steps such as washing, heat pre-treatment, potential digestion,stringency wash and other incubation steps can be performed. Therelatively short distance between the block and the carrier when theblock is a second distance results in fast heating and/or cooling of theapplied processing liquid to the specified temperatures in differentprocessing steps, avoids problems with temperature gradients, andsecures the use of small volumes of processing liquids.

The block can also be in a third or further distance(s), dependent onthe required liquid volume for the specific processing step. This givesthe apparatus according to the invention great flexibility.

An efficient mixing of liquids applied to the carrier can be obtained byrepeating one or more of the x/y/z movements a number of times. Suchmixing can increase the reaction rate by providing active movement ofliquid components applied to the sample. It should be understood thatthe mixing may be a so-called chamber-mixing independent of the carrieror a carrier micro-mixing.

More than one processing liquid can also be applied to the carrier orblock and subsequently be mixed in the apparatus. According to oneembodiment the liquids can be mixed in the bottom of the chamber, i.e.not in direct contact with the sample, where after a movement of theblock to a first processing position spreads the mixed liquid over thebiological sample creating a thin film.

FIGS. 3 a and 3 b schematically illustrate an embodiment of theapparatus 100 comprising a lid 114. The lid 114 is configured to bearranged at said first structure 109 to enclose said carrier 103 andsaid second structure 110 during processing of said biological sample101 arranged on said first planar surface 102.

As illustrated in FIGS. 3 a and 3 b, the embodiment of the apparatus 100may comprise a reservoir 118 arranged at a surface 119 of said lid 114that faces said second structure 110. The reservoir 118 may be tocontrol the humidity within the processing chamber 108 during processingof said biological sample 101. The reservoir 118 may be configured as anelongated reservoir 118 comprising an absorbing material.

Humidity can also be controlled by providing a humidity reservoir, forexample a reservoir in one or more of the block, platen or wall.

FIG. 10 a shows an embodiment of a processing apparatus 100′ forprocessing of two biological samples arranged on two carriers 103, 103′.As is shown in the figure, the block 110′ has two planar surfaces 104,104′. An inlet/outlet 122′ is also shown.

In FIG. 10 b it is shown that an amount of processing liquid 105, 105′can be applied to the planar surfaces 104, 104′ of the block 110′, whenthe block is in upper position and having the planar surfaces 104, 104′facing away from the first structure 109, 109′. In the figure it is alsoshown, when the block 110′ is in a processing position, i.e. when theblock 110′ has been turned around to a position when the planar surfaces104, 104′ are facing the first structure 109, 109′ and the carriers 103,103′ and when the block 110′ has been inserted into the first structure109, 109′).

FIG. 11 a shows a multiprocessing apparatus 129 comprising 12 processingchambers 108 having first structures 109 and blocks 110, and 12 carriers103. The figure shows 12 blocks 110 but it should be understood that oneblock could be designed to cover all 12 carriers or that a block couldbe designed to cover a pre-determined number of carriers.

FIGS. 11 b and 11 c show an embodiment of a multiprocessing apparatus129. In this embodiment, the carriers 103 are mounted on a carrierholder 130, e.g. a rack 130, whereby all the carriers 103 can be movedsimultaneously. The carriers 103 may be moved in the directions of thearrow. FIG. 11 c shows the supply of an amount of processing liquid 105to each carrier 103, by means of a supply means 131 connected to aprocessing liquid source (not shown). In embodiments, the number ofsupply means 131 corresponds to the number of carriers 103, but itshould be understood that one supply means could be configured to supplyliquid to all of the carriers. In such embodiments, the supply meanscould be part of a movable robot means.

FIG. 12 shows part of an embodiment of a processing apparatus forprocessing two biological samples arranged on carriers 103, 103′. Herethe two tissue sections are facing each other and they are in contactwith the same liquid during processing. This could be advantageous e.g.when one of the slides is a control slide. In other embodiments, thesample carriers comprise biological samples to be processed by the sameprocessing liquid. In yet other embodiments, one of the sample carriersis blank, i.e. it does not contain a sample, whereby the blank samplecarrier when arranged at the lower bottom plate 111 or the block 110 mayserve to protect the lower bottom plate 111 or the block 110,respectively, from being contaminated with processing liquid. In theshown embodiment, the lower bottom plate 111 comprises a lower heatingelement 124 and the block 110 comprises an upper heating element 124′.

FIGS. 13-15 schematically show parts of an embodiment of the processingapparatus according to the invention.

FIG. 13 shows schematically in perspective a side view of a samplecarrier 103 with a biological sample 101 and a block 110. As illustratedin the figure, the block 110 comprises a planar surface 104 also calleda sample side or second planar surface, an upper side 140, a left side142, a right side 144, a back side 146. A handle 148 may be arranged atan upper part of the block 110. The block 110 comprises further sideedges 150 a, 150 b, 150 c, 150 d, and a lower edge 152.

FIG. 14 shows schematically a side view of the sample carrier 103 andblock 110. As shown, the planar surface 104 comprises a planar elevation104 a arranged to cover at least a part of the sample 103. The planarsurface 104 may also comprise a second elevation 104 b configured tofunction as a sealing between the block 110 and the sample carrier 103when the block 110 is arranged in a processing position. As illustratedin FIG. 14, the block 110 may in a lower part comprise a recessconfigured to provide a reservoir 121 when the block 110 is arranged ina processing position.

FIG. 15 schematically shows a cross section of the first structure 109and the bottom plate 111. The first structure 109 comprises a back side112 a, a left side wall 112 b, and a right side wall 112 c. The bottomplate 111 may comprise a recess 113 configured to carry the samplecarrier 103. The recess and the bottom plate may comprise an aluminiumbottom. Embodiments may be provided with a sealing 133 arranged betweenthe first structure 109 and the bottom plate 111. The sealing 133 may bea rubber strip or the like. FIG. 15 also shows a lower/bottom inner wall154 of the processing chamber, a back screw 156 configured to secure theblock at a first position and a fastening means 158 such as a hook orthe like. By mean of which fastening means 158, the processing chambermay be removable fastened in the processing apparatus.

FIG. 16 shows an embodiment of a block 110. The block 110 comprises asecond planar surface 104 comprising a planar elevation 104 a arrangedto cover at least a part of a biological sample 101 during processingwhen said second planar surface 104 is at said second distance from saidfirst planar surface 101. The elevation 104 a may have the shape of acover glass and at least have such a shape that substantially covers thetarget area. The second planar surface 104 may also be provided with asecond elevation 104 b in a front part thereof. The second elevation 104b may be planar and may be arranged to function as a sealing or barrier.

A sealing 133 may be arranged at the outer boundary of said planarelevation 104 a. The sealing 133, e.g. a rubber strip, secures that theapplied amount of liquid is retained within a space defined by saidsealing 133, said planar elevation 104 a, and said part of saidbiological sample 101. Outside the space defined by the sealing 133,said planar elevation 104 a, and said part of said biological sample 101is a humidity channel to the liquid reservoir 121, whereby a relativehigh humidity can be provided during processing.

FIG. 17 shows schematically an embodiment of an inventive system. Thesystem comprises a processing apparatus 100. A control unit 200 may beconfigured to control the operation of components of the system. Forexample, the control unit 200 may be configured to control pumping means230, such as a pump, and robot means 220 to supply a processing liquidfrom a liquid source 126. Further, the control unit controls theoperation of the processing apparatus 100 and may interact with aninput/output interface 210, such as a display.

FIGS. 18 a, 18 b and 18 c show schematically embodiments of a block 110.As illustrated, the block comprises a planar elevation 104 a in theplanar surface 104. Embodiments of the block may comprise a frontsupport 160 and a back support 162 arranged to provide a humiditychannel 132 around the biological sample when the block 110 is in aprocessing position. The supports 160, 162 may be arranged to provide adistance of e.g. 1 mm between the planar surface 104 and the samplecarrier when the block is in the processing position. The supports 160,162 can also suitably be positioned on the planar surface 104 such thatit will rest on the surface of the first planar surface, e.g. a slide.

When a block according to the embodiment shown in FIG. 18 a is in aprocessing position, the humidity channel 132 will be open in the frontpart due to the recess 166 between the front supports 160 and theelevation 104 a.

On the other hand, when a block according to the embodiment shown inFIG. 18 c is in a processing position, the humidity channel 132 will beclosed which will reduce evaporation of processing liquid. An embodimentof the block according to FIG. 18 b will provide a semi-open (orsemi-closed) humidity channel 132.

In a front part of the block, the block is provided with a recess 164for supply of processing liquid. The recess 164 may have different shapeand size, but should be designed to facilitate the supply of processingliquid when the block is in the processing position.

FIG. 19 schematically shows an embodiment of an apparatus for processinga biological sample according to the invention, in the embodiment theside wall 112 c is removed. The block 110 in FIG. 19 corresponds to theblock 110 shown in FIG. 18 a, but it should be understood that the blockcould be of another type or have another design.

As illustrated in FIG. 19, the bottom plate 111 may be provided with achannel 168 surrounding the part of the bottom plate 111 supporting thecarrier 103. As illustrated, the channel 168 may be in fluidcommunication with the inlet/outlet 122 by means of which inlet/outlet122 liquid may be supplied to or withdrawn from the channel 168.Further, the channel 168 may have one or more inclined side walls 169.

It should be understood that the channel 168 may be arranged in theblock 110 and/or in the bottom plate 111 as described with reference toFIG. 19.

An embodiment of the method for processing biological samples comprisesthe steps of:

-   -   lifting up a block of a processing chamber to an upper position;    -   adding an amount of a processing liquid, e.g. a probe buffer,        either manually or automatically, to the biological sample to be        treated. This may be done after a pre-treatment procedure;    -   moving the block to a processing position, whereby the applied        processing liquid is distributed over the biological sample; and    -   processing the biological sample during a predefined time period        during an optional heating and/or cooling.

Another embodiment of the method for processing biological samplescomprises the steps of:

-   -   lifting up the block of a processing chamber to an upper        position;    -   rotating the block:    -   adding an amount of a processing liquid, e.g. a probe buffer,        either manually or automatically, to the block for further        application to the biological sample. This may be done after a        pre-treatment procedure;    -   rotating the block back;    -   moving the block to a processing position, whereby the applied        processing liquid is distributed over the biological sample; and    -   processing the biological sample during a predefined time period        during an optional heating and/or cooling procedure.

Another embodiment of the method for processing biological samplescomprises the steps of:

-   -   lowering a block to lowermost processing position;    -   adding an amount of a processing liquid, e.g. a probe buffer,        either manually or automatically, to the biological sample to be        treated. The processing liquid is distributed by gravity and/or        capillary forces. This may be done after a pre-treatment        procedure; and    -   processing the biological sample during a predefined time period        during an optional heating and/or cooling procedure.

The following is an example of IHC and FISH procedures performed usingthe present invention providing good results. The apparatus of theinvention is used from the process steps of baking until cover slippingupon paraffin embedded tissues. The examined tissues have been fixed inneutral buffered formaldehyde from 6 to 72 hours. The tissues consistof: tonsil, liver, mamma carcinoma, medullary thyroid cancer, coloncancer, melanoma metastasis, colon, prostate, cerebellum, kidney andpancreas.

For the IHC, volumes of 10 mL were used in the deparaffination/TargetRetrieval, Wash Buffer and H₂O process steps. For reagents such asperoxidase, antibodies, detection system, hematoxylin volumes of 50 ′Lwere used. The process temperature is 37° C. unless otherwise specified.

Same or similar results were obtained with volumes of 20 μL, 30 μL, 40μL and larger.

Baking was performed at 60° C. 10 min; combined deparaffination andTarget Retrieval (S1700, Dako) at 95° C. 10 min; wash (Wash Buffer 1×,S3306, Dako) for 5 min; peroxidase block (S2023, Dako) 5 min; wash 5min; monoclonal mouse anti-human cytokeratin (M3515, Dako) 10 min; wash5 min; detection (K5007); rinse (Wash Buffer); wash 5 min; chromogenDAB+ (K3468, Dako) 2×5 min; wash 5 min; counterstaining with Hematoxilin(S3301) for 5 min; rinse Wash Buffer; rinse with H₂O. The process timewas about 90 minutes. Mounting was then performed in Faramount MountingMedium (S3025, Dako).

For FISH volumes of 9 mL (brick lifted 3 mm) were used in thedeparaffination/Pre-Treatment, Wash Buffer, Stringent Buffer and H₂Oprocessing steps. For the probe buffer a volume of 30 μL was used (brick0 mm position). In baking and drying steps the brick was elevated 10 mm.The process temperature was 37° C. unless otherwise specified.

Same or similar results were obtained with volumes of 20 μL, 30 μL, 40μL and larger.

Baking was performed at 60° C. 10 min; combined deparaffination andTarget Retrieval (S1700, Dako) at 95° C. 10 min; wash (Wash Buffer 1×,S3306, Dako) for 5 min; wash 5 min (Wash Buffer 1×, K5331, Dako); PepsinRTU (K5331, Dako) 4 min; wash 5 min (Wash Buffer 1×, K5331, Dako); washwith H₂O 2 min; drying (dehydration) 5 min; 400 μL H₂O to liquidreservoir; 30 μL HER2 FISH probe (K5331, Dako) at 82° C. 5 min and at45° C. O/N (about 16 h); Stringency Wash (K5331, Dako) 65° C. 10 min;wash 5 min; wash with H₂O 2 min, drying (dehydration) 5 min. The processtime until hybridization step was about 60 minutes and the process timefrom hybridization was stopped until mounting for about 30 min. Themounting was then performed in 15 μL Antifade Mounting Medium (K5331,Dako).

1-41. (canceled)
 42. A method for processing a biological sample byperforming one or more processing steps, comprising: inserting a samplecarrier holding the biological sample into a container comprising abottom plate and at least three side walls; positioning a block withinthe container such that a sample side surface of the block is arrangedsubstantially parallel to the bottom plate of the container and thecarrier; forming a processing chamber between the container and theblock, which provides a variable confined volume that is variable bychanging a distance of the sample side surface of the block from thebottom plate of the container; supplying an amount of a liquid to thebiological sample for the one or more processing steps; orienting thecontainer such that the bottom plate forms an angle greater than 0degrees and less than 90 degrees with a horizontal plane on which thecontainer is disposed; and draining a portion of the liquid from theprocessing chamber via a gravitational drain formed in the bottom plate.43. The method of claim 42, wherein supplying an amount of the liquid tothe biological sample comprises releasing the liquid to flow from aliquid supply arranged at an upper end of the processing chamber throughthe processing chamber at a rate determined by gravity and capillaryforces.
 44. The method of claim 42, further comprising mixing the liquidwithin the processing chamber by moving the sample side surface relativeto the sample carrier.
 45. The method of claim 42, further comprisingpositioning the block such that a planar elevation formed on the sampleside surface covers at least a part of the biological sample.
 46. Themethod of claim 45, further comprising sealing the outer boundary of theplanar elevation such that liquid supplied to the biological sample isretained within a space defined by the outer boundary of the planarelevation and the part of the biological sample.
 47. The method of claim42, adjusting the distance of the sample side surface of the block fromthe sample carrier using an automatic distance changing apparatus. 48.The method of claim 42, wherein the angle is between 30 degrees and 60degrees, and more preferably approximately 45 degrees.
 49. The method ofclaim 42, further comprising installing a lid configured to be arrangedon the container to enclose the sample carrier and the block duringprocessing of the biological sample.
 50. The method of claim 49, furthercomprising controlling the humidity within the processing chamber duringprocessing of the biological sample by way of a reservoir arranged at asurface of the lid that faces the block.
 51. The method of claim 42,further comprising controlling the processing temperature according tothe one or more processing steps.
 52. The method of claim 51, whereincontrolling the processing temperature includes heating and cooling. 53.The method of claim 42, wherein the processing is in-situ-hybridization,immunohistochemistry, or special stain procedure.
 54. The method ofclaim 42, wherein the one or more processing steps include one or moreof baking, fixation, dehydration, deparaffinization, target retrieval,pretreatment, static wash, dynamic wash, digestive treatment,denaturation, hybridization, mounting, coverslipping, H&E staining,special staining, IHC staining, and counterstaining.
 55. The method ofclaim 42, wherein the one or more processing steps are all performedwhile the sample carrier is in one position.
 56. A method for processinga biological sample by performing one or more processing steps,comprising: inserting a sample carrier holding the biological sampleinto a container comprising a bottom plate and at least three sidewalls; positioning a block within the container such that a sample sidesurface of the block is arranged substantially parallel to the bottomplate of the container and the carrier; forming a processing chamberbetween the container and the block, which provides a variable confinedvolume that is variable by changing a distance of the sample sidesurface of the block from the bottom plate of the container; andreleasing an amount of a liquid to the biological sample for the one ormore processing steps.
 57. The method of claim 56, further comprisingdraining a portion of the liquid from the processing chamber via agravitational drain formed in the bottom plate.
 58. The method of claim56, further comprising orienting the container such that the bottomplate forms an angle greater than 0 degrees and less than 90 degreeswith a horizontal plane on which the container is disposed.